The field of the invention is cell death.
Programmed cell death is a physiological process that has been conserved through evolution (Ellis, Ann. Rev. Cell Biol. 7:663-698, 1991; Raff, Nature 356:397-400, 1992). Genetic analyses of the cell-death process in C. elegans have defined a genetic pathway for programmed cell death (reviewed by Horvitz et al., Cold Spring Harbor Symposia on Quantitative Biology LIX, 377-385, 1994). Mutations in three genes, ced-9, ced-4, and ced-3 (ced, cell death abnormal), affect, most if not all, of the 131 somatic cell deaths that occur during the development of the C. elegans hermaphrodite (Sulston and Horvitz, Dev. Biol. 56:110-156, 1977; Sulston et al., Dev. Biol. 100:64-119, 1983). Loss-of-function (lf) mutations in ced-3 or ced-4 result in the survival of cells that normally die, indicating that these genes are required for the killing process (Ellis and Horvitz, Cell:44:817-829, 1986). ced-9, by contrast, is a negative regulator of programmed cell death. A gain-of-function (gf) mutation in ced-9 prevents most if not all programmed cell deaths, and loss-of-function (lf) mutations in ced-9 cause embryonic lethality as a consequence of ectopic cell death (Hengartner et al., Nature 356:494-499, 1992). This lethality is suppressed by loss-of-function mutations in ced-3 or ced-4, indicating that ced-3 and ced-4 act downstream of, or in parallel to, ced-9 (Hengartner et al., Nature 356:494-499, 1992). ced-4 is likely to act upstream of ced-3, since cell death induced by ced-4 overexpression is greatly reduced in the absence of ced-3 activity (Shaham and Horvitz, Genes Dev. 10:578-591, 1996). The ced-9, ced-4, and ced-3 central cell-death machinery is thought to be regulated by cell-type-specific regulators, which includes the cell-death specification genes ces-1 and ces-2; these two genes specify the life-versus-death decisions of a subset of cells, including the sisters of the NSM neurons in the pharynx (Ellis and Horvitz, Development 112:591-603, 1991).
The genetically established interactions among ced-9, ced-4, and ced-3 may reflect direct physical interactions of the protein products of these genes. The CED-9 protein binds to the CED-4 protein (Spector et al., Nature 385:653-656, 1997; Chinnaiyan et al., Science 275:1122-1126, 1997; Wu et al., Science 275:1126-1129, 1997; James et al., Curr. Biol. 7:246-252, 1997; Ottilie et al., Cell Death and Differentiation 4:526-533, 1997), which, in turn, can bind to the CED-3 protein (Chinnaiyan et al., Science 275:1122-1126, 1997; Wu et al., J. Biol. Chem. 272:21449-21454,1997). Furthermore, the interaction of CED-4 with CED-3 appears to lead to the activation of CED-3 and the initiation of cell death (Seshagiri and Miller, Curr. Biol. 7:455-460, 1997; Chinnaiyan et al., Nature 388:728-729, 1997; Wu et al., J. Biol. Chem. 272:21449-21454,1997).
ced-9 and ced-3 have mammalian counterparts also shown to be involved in programmed cell death. ced-9 encodes a protein structurally and functionally similar to the mammalian cell-death inhibitor Bcl-2 (Hengartner and Horvitz, Cell 76:665-676, 1994), the prototype of a family of Bcl-2-like molecules that act as regulators of cell death in mammals (reviewed by White, Genes Dev. 10:1-15, 1996; Rinkenberger and Korsmeyer, Curr. Op. Gen. Dev. 7:589-596, 1997). CED-3 is a member of a family of invertebrate and mammalian cysteine proteases, collectively called caspases, that are cell-death effectors acting mainly downstream of Bcl-2-like cell-death regulators (reviewed by Fraser and Evan, Cell 85:781-784, 1996; Nicholson and Thomberry, Trends Biochem. Sci. 22:299-306, 1997).
Recently, a new group of mammalian cell-death activators has been identified. These proteins, which include Bik, Bid, Harakiri, and Bad, interact with Bcl-2-like proteins and can induce cell death when overexpressed (Yang et al., Cell 80:285-291, 1995; Boyd et al. Oncogene 11:1921-1928, 1995; Han et al., Mol. Cell. Biol. 16:5857-5864, 1996; Wang et al., 1996; Inohara et al., EMBO J. 16:1686-1694, 1997; Zha et al., J. Biol. Chem. 272:24101-24104, 1997; Kelekar et al., Mol. Cell. Biol. 17:7040-7046, 1997; Ottilie et al., J. Biol. Chem. 272:30866-30872, 1997). The amino acid sequences of these cell-death activators are dissimilar, except for a nine amino acid stretch similar to one of the four Bcl-2 homology (BH) domains, the BH3 domain, and particularly similar to the BH3 domain of the Bcl-2-like cell-death activators Bax and Bak (Chittenden et al., EMBO J. 14:5589-5596, 1995; 1995; Han et al., Genes Dev. 10:461-477,1996 1996b; Zha et al., J. Biol. Chem. 271:7440-7444, 1996; Hunter and Parslow, J. Biol. Chem. 271:8521-8524, 1996). As in the cases of Bax and Bak (Chittenden et al., EMBO J. 14:5589-5596, 1995; Han et al., Genes Dev. 10:461-477,1996), the BH3 domains of this new group of cell-death activators are important both for their interaction with Bcl-2-like molecules and for their ability to induce cell death (Wang et al., Genes Dev. 10:2859-2869, 1996; Inohara et al., EMBO J. 16:1686-1694, 1997; Zha et al., J. Biol. Chem. 272:24101-24104, 1997; Kelekar et al., Mol. Cell. Biol. 17:7040-7046, 1997; Ottilie et al., J. Biol. Chem. 272:30866-30872, 1997).
It would be useful to identify and clone additional genes in the C. elegans cell death pathways. Due to the conservation between nematode and mammalian cell death pathways, identification of such genes and their encoded proteins could allow detection of therapeutic targets, therapeutic compounds, and novel cell death genes.
We have discovered and cloned a new C. elegans cell death gene, egl-1 (egl, egg-laying defective) that encodes a protein that interacts with CED-9 and that contains a region similar to the BH3 domains of BH3-containing cell-death activators. Gain-of-function mutations in egl-1, such as egl-1(n1084 n3082), cause the two HSN neurons, which are required for egg laying, to inappropriately undergo programmed cell death; these mutants were identified in screens for egg-laying defective (Egl) mutations (Trent et al., Genetics 104:619-647, 1983). By isolating a dominant suppressor of the egl-1 Egl phenotype, we identified a loss-of-function mutation in the egl-1 gene, egl-1(n1084 n3082). This mutation prevents not only the ectopic deaths of the HSNs but most if not all normally occurring programmed cell deaths, indicating that egl-1 is a cell-death activator and encodes a component of the general cell-death machinery in C. elegans. 
In the first aspect, the invention features substantially pure nucleic acid encoding EGL-1 polypeptide. Such nucleic acid is defined by its ability to complement any of the egl-1(n1084 n3082) mutations provided herein or by the ability to suppress mutations in egl-1(n1084). Preferably, specifically excluded is the exact wild-type nucleic acid sequence provided at the ced(n3082) map position in the C. elegans Genome Consortium database on May 28, 1997; conservative substitutions of this sequence are, however, preferably included. In a related aspect, the invention features egl-1 nucleic acids which have deletions. Fusions of additional nucleic acids to the egl-1 gene are also included, as are fragments sufficient for use as primers, probes, or synthesis of epitopes for antibody preparation. Homologs of C. elegans egl-1 from other species (and fragments, fusions, deletions, and other mutations therein) are also a related aspect of the invention. Homologs are defined as having at least 50%, preferably 90% identity over at least 100 base pairs, or as being able to complement at least one egl-1(n1084 n3082) allele and having at least 20% identity over the entire gene. Identity is preferably determined using the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wis. 53705, on the default settings.
In a related aspect the invention provides polypeptide encoded by the egl-1 gene. Proteins encoded by egl-1 nucleic acids which are fragmented, deleted or fused to other sequences are also included. For example, the BH3 domain (and nucleic acids encoding the same).
In another aspect, the invention features an antibody which specifically binds a protein encoded by the egl-1 gene. By xe2x80x9cspecifically bindsxe2x80x9d is meant capable of binding with a kd of at least 10-8M.
In another aspect, the invention features a nematode having a mutation in the egl-1 gene. Preferably, the mutation is genetically engineered or is a suppressor of the egl-1 Egl phenotype.
In yet another aspect, the invention features a method for identifying a compound or gene which affects cell death, said method comprising exposing a nematode having either a egl-1(n1084 n3082) or other egl-1 mutation to the compound or nucleic acid and looking for amelioration of the phenotype caused by the gene. In a related aspect the invention features a method of identifying novel cell death genes or alleles by looking for genetic suppressors of either egl-1 mutations or mutations which are egl-1(n1084 n3082)-like mutations mapping within a map unit of egl-1.
In yet other aspects of the invention, methods for detecting proteins which interact with EGL-1 and methods for purifying non-C. elegans homologs of EGF-1 are also provided.
By xe2x80x9cmodulating cell deathxe2x80x9d or xe2x80x9caltering cell deathxe2x80x9d is meant increasing or decreasing the number of cells which undergo programmed cell death in a given cell population. Preferably, the cell population is selected from a group including T-cells, neuronal cells, fibroblasts, or any other cell line known to undergo apoptosis in a laboratory setting (e.g., the baculovirus infected insect cells). It will be appreciated that the degree of modulation provided by an EGL-1 polypeptide or modulating compound in a given assay will vary, but that one can determine the statistically significant change in the level of cell death which identifies an EGL-1 polypeptide or a compound which modulates the egl-1 gene or the EGL-1 polypeptide.
By xe2x80x9cinhibiting cell deathxe2x80x9d is meant any decrease in the number of cells which undergo programmed cell death relative to an untreated control. Preferably, the decrease is at least 25%, more preferably the decrease is 50%, and most preferably the decrease is at least one-fold.
By xe2x80x9cpolypeptidexe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% homology to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably over the entire nucleotide sequence of the referenced sequence.
Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By a xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant an EGL-1 polypeptide which has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, EGL-1 polypeptide. A substantially pure EGL-1 polypeptide may be obtained, for example, by extraction from a natural source (e.g., a fibroblast, neuronal cell, or lymphocyte cell); by expression of a recombinant nucleic acid encoding an EGL-1 polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., those described in column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.
By xe2x80x9csubstantially pure nucleic acidxe2x80x9d is meant nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant nucleic acid which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. Further included are RNA molecules encoded by egl-1 and nucleic acid sequences which are antisense.
By xe2x80x9ctransformed cellxe2x80x9d is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) an EGL-1 polypeptide.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic nematodes or mammals (e.g., rodents such as rats or mice) and the DNA (transgene) is inserted by artifice into the nuclear genome or by an extrachromosomal array.
By xe2x80x9ctransformationxe2x80x9d is meant any method for introducing foreign molecules into a cell. Microinjection, lipofection, calcium phosphate precipitation, retroviral deliver, electroporation and biolistic transformation are just a few of the teachings which may be used. For example, biolistic transformation is a method for introducing foreign molecules into a cell using velocity driven microprojectiles such as tungsten or gold particles. Such velocity-driven methods originate from pressure bursts which include, but are not limited to, helium-driven, air-driven, and gunpowder-driven techniques. Biolistic transformation may be applied to the transformation or transfection of a wide variety of cell types and intact tissues including, without limitation, intracellular organelles (e.g., and mitochondria and chloroplasts), bacteria, yeast, fingi, algae, animal tissue, and cultured cells.
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., an EGL-1 polypeptide, a recombinant protein or a RNA molecule).
By xe2x80x9creporter genexe2x80x9d is meant a gene whose expression may be assayed; such genes include, without limitation, xcex2-glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), and xcex2-galactosidase.
By xe2x80x9cpromoterxe2x80x9d is meant minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
By xe2x80x9cconserved regionxe2x80x9d is meant any stretch of six or more contiguous amino acids exhibiting at least 30%, preferably 50%, and most preferably 70% amino acid sequence identity to EGL-1.
By xe2x80x9cdetectably-labelledxe2x80x9d is meant any means for marking and identifying the presence of a molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule. Methods for detectably-labelling a molecule are well known in the art and include, without limitation, radioactive labelling (e.g., with an isotope such as 32p or 35S) and nonradioactive labelling (e.g., chemiluminescent labelling, e.g., fluorescein labelling).
By xe2x80x9cpurified antibodyxe2x80x9d is meant antibody which is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody, e.g., an EGL-1 specific antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced protein or conserved motif peptides and standard techniques.
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody which recognizes and binds a protein but which does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, which naturally includes protein.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.